Chromatographic separation systems are often used to separate a protein of interest from a solution containing the protein of interest and one or more contaminants. Typically, a chromatographic protein purification system comprises a plurality of chromatography columns, each having different properties, through which the solution is passed in a particular order. For example, a protein purification system may use some or all of the following 4 types of columns in turn:
affinity columns, desalting columns, ion exchange columns and gel filtration columns. Such systems can be controlled by a computer or micro processor provided with suitable software.
In order to purify a sample containing the protein of interest an operator would choose an affinity column containing a medium provided with a ligand to which the protein of interest specifically, but reversibly, binds. The sample containing the protein of interest is applied to the column under conditions that favour binding of the protein of interest to the ligand. Once the whole of the sample containing the protein of interest has been passed through the medium, the column is washed of unbound material and then the protein of interest is eluted from the medium by changing the buffer passing through the column to a buffer, for example a buffer with a high salt concentration, which breaks the bond between the protein of interest and the ligand. The presence of the protein of interest in the buffer eluted from the column is detected by a detector such as a UV-detector which detects changes in the UV absorbance at a specific wavelength, e.g. 280 nm or 254 nm, in the elution buffer leaving the column, which absorbance provides a rough measurement of total protein eluting in a given fraction. When eluting a column the fluid is collected in a fraction collector. The software monitors the UV-detector output which indicates when proteins including the protein of interest are leaving the column and records in memory to which fraction this corresponds. After the run through the first column is completed the operator checks the UV curve and manually collects the fractions of interest containing the protein. The manually collected fractions are manually pooled and transferred to the next column. If a high salt concentration elution buffer was used, then the elution buffer containing the protein of interest can be desalted by passing it through a desalting column. The elution solution outputted from the desalting column is monitored by a detector, for example, a UV-detector and the volume of desalted solution containing the protein of interest leaving the desalting column is directed to a fraction collector. The operator manually pool fractions of interest and transfer the pooled fractions to the next column. This volume of desalted solution may still contain contaminants and it may be necessary to use an ion-exchange column to remove some of them. If so, then the operator passes the volume of desalted solution containing the protein of interest through an ion exchange chromatography column which contains a medium which allows binding of the protein of interest to it. Once the volume of desalted sample containing the protein of interest has been applied to the column and the protein of interest reversibly bound to the column medium, the column can be washed to remove unbound substances. The bound substances, which include the protein of interest and possibly some contaminants, can then be removed from the medium by changing to elution conditions which are unfavourable for the ionic bonding of the protein of interest. The elution solution exiting the ion-exchange column is monitored by a detector, for example, a UV-detector and the volume of elution solution containing the protein of interest leaving the ion exchange column is collected in a fraction collector. This volume of ion-exchanged solution may still contain contaminants, e.g. aggregates of protein molecules, and it may be necessary to use a gel filtration column to remove some of them. If so, then the operator passes the volume of ion-exchanged solution containing the protein of interest through a gel filtration column. The filtered solution leaving the gel filtration is collected in a fraction collector. If the choices of medium and elution conditions in each column have been correctly chosen to favour separation of the protein of interest from the solution, then by now the protein of interest should be the substance having the highest concentration in the volume of ion-exchanged solution passing through the gel filtration column.
The above method of purifying a protein is time-consuming and laborious. It requires frequent operator actions such as evaluating the result from each column and moving fractions of elution solution from column to column.
In order to reduce these problems automated systems have been produced in which a detector signal is monitored by a computer and the collection of a fraction is started when the detector signal rises above a certain value (which corresponds to the concentration of proteins in the solution passing the detector rising above a threshold) and the collection of the fraction is stopped when the detector signal drops below a certain value (which corresponds to the concentration of proteins in the solution passing the detector falling below a threshold). In order to achieve this, the software is provided with monitoring subroutines called “Watch functions” for initiating certain actions when the absorbance detected by the UV detector passes a threshold set by the operator. A “Level Greater Than X1” watch function causes the computer to divert the flow passing the UV detector to a new container in the fraction collector and to note the number of the fraction collector that the fluid is being diverted to when the signal for the detector parameter corresponding to the absorbance level detected by the UV detector passes the value X1. It also starts a “Level Less Than Y1” watch function. A “Level Less Than Y1” watch function causes the computer to divert the flow passing the UV detector from the current container in the fraction collector to a new container (or to waste) and restarts a “Level Greater Than X1” watch function when the absorbance detected by the UV detector falls below the level Y1. While these automated systems overcome some of the problems caused by operator error, the rules which the computer follows in order to start and stop the collection of fractions have hitherto been unsophisticated. For example, the rule of starting collection when a detector signal rises above a first threshold and stopping collection when it falls below a second threshold has not been robust enough to ensure a proper collection of all the different peak shapes that have to be recognised by the software. FIG. 1 shows some examples of UV-absorbance curves which proteins can produce when leaving a column. FIG. 1 shows at A a curve with single peak which could represent a single protein leaving the column. FIG. 1 shows at B a composite curve formed of two overlapping peaks with a trough or valley between the peaks which could present two similar proteins leaving the column. Here the detector level at the lowest point of the valley is greater than half the maximum value of the first peak. It may be desired to collect these two peaks as one fraction. FIG. 1 shows at C, another composite curve formed of two overlapping peaks with a deeper valley between the peaks with a value less than half of the maximum value of the first peak which could represent two less similar proteins leaving the column. It is often desirable to collect these two peaks as separate fractions. FIG. 1 shows at D another composite curve formed of two peaks which could represent two similar proteins where the first protein is less abundant than the second protein. FIG. 1 shows at E a peak followed by a stable plateau. It is often desirable to collect the peak and plateau as separate fractions. Prior art systems which use “Level Greater Than X1” and “Level less Than Y1” can have problems separating the peaks shown in FIG. 1 at B-E.
Additionally, UV detector noise and instabilities such as instrument drift over time can lead to spurious output signals which can activate a “Level Greater Than X1” function inadvertently.
This lack of robustness means that manual intervention is still required during the separation of proteins of interest from contaminants.